DESCRIPTION (from the application): The long-term goal of this research is to understand the molecular mechanisms that govern the formation of cartilage and bone, and specifically, the molecular basis for commitment of progenitor cells to the chondrocyte differentiation pathway. The underlying hypothesis is that the entry of stem cells into the chondrogenic lineage is accompanied by changes in gene expression that, in turn, are controlled by specific transcription factors. We here propose to identify such transcription factors using the murine homeobox gene Hoxc-8 as a paradigm. Research in my and other laboratories demonstrates that Hox genes play critical roles in development of the skeleton, by regulating proliferation and/or differentiation of skeletogenic cells. Our Preliminary Results provide strong evidence that Hoxc-8 (and Hoxd-4) regulate proliferation of chondrocyte precursor cells in vivo. In addition, we found that distinct cis-regulatory regions control the expression of the Hoxc-8 gene in somites and in the chondrogenic lineage. This implies that with the commitment of mesodermal cells to the chondrocyte pathway, different regulatory elements, and hence, different transcriptional regulatory factors become activated. The guiding postulate for this proposal is that the transcription factors that accompany the switch in Hoxc-8 expression are important regulators of chondrocyte progenitor cell commitment. The identification of such transcription factors will provide novel insights into the molecular regulation of chondrogenic precursor cells. Specifically, this research will (1) investigate the cell type-specificity of Hoxc-8 expression in the developing cartilage by in situ hybridization and immunohistochemistry; (2) identify the cartilage-specific regulatory elements in the Hoxc-8 gene in vivo through a deletion analysis of the Hoxc-8 gene promoter by LacZ reporter gene activity in transgenic mice; (3) define the relevance of specific sites by site directed mutagenesis followed by electrophoretic mobility shift assays in vitro and in vivo assays in transgenic mice; and (4) identify and isolate transcription factors that bind to the cartilage element in the Hoxc-8 gene by gel purification, sequencing and cDNA cloning. We will then investigate the expression and functional role of these proteins. A better understanding of the molecular biology of chondrogenic progenitor cells will form the basis for future research into techniques for stem cell manipulations in vitro or in vivo as potential therapeutic approaches in the regeneration and repair of cartilage in the healing of fractures or in treatment of diseases, such as arthritis.